西花蓟马FoccOBP3的分子克隆、序列分析及表达特征

Cloning, Sequence Analysis and Expression Characteristics of FoccOBP3 in Western Flower Thrips Frankliniella occidentalis

鉴定昆虫嗅觉相关蛋白基因并对其序列和时空表达进行研究,可为阐明昆虫与寄主间的化学通讯机制提供依据。本文新克隆并鉴定获得一个西花蓟马OBP基因FoccOBP3(Gen Bank登录号:MT682352),c DNA序列全长1226 bp,读码框全长432 bp,编码143个氨基酸残基。氨基酸序列中六个保守的半胱氨酸位点排列方式为C_1-X_(26)-C_(2)-X_(3)-C_(3)-X_(37)-C_4-X_(10)-C_(5)-X_8-C_(6),完全符合典型气味结合蛋白所具有的六个保守的半胱氨酸位点结构模型。氨基酸序列中有5个亲脂性区域,其中第62~75位的氨基酸残基形成明显的亲脂性口袋。预测该蛋白分子量为15.50 kD,等电点为5.24,N-末端包含21个氨基酸组成的信号肽序列。FoccOBP3与横缝茧蜂Diachasmaalloeum的DallGOBP(Gen Bank登录号为XP_015125081.1)、赤拟谷盗Triboliumcastaneum的TcasPBP(Gen Bank登录号为XP_015835846.1)和TcasOBP07(Gen Bank登录号为EFA04593.1)的氨基酸序列一致性较高。FoccOBP3与埋葬甲虫Nicrophorusvespilloides的NvesGOBP(Gen Bank登录号为XP_017781594.1)聚在最小的一个分支,表明与该基因亲缘关系最近。Focc OBP3蛋白含有组成α-螺旋的氨基酸残基127个,形成β-折叠的氨基酸残基58个,形成β-转角的氨基酸残基15个。Focc OBP3蛋白骨架是由6个α-螺旋和连接这些螺旋的回折构成,其中5个α-螺旋形成一个结合口袋。FoccOBP3在西花蓟马1龄若虫中高表达,其次是羽化5d的雌虫,其余发育阶段的表达量均较低;且在雌、雄性成虫中的表达量亦有差异,除了羽化10d的,羽化1、5、15d雌虫的表达量均比同发育阶段雄虫的高。该基因在西花蓟马成虫触角中高表达,其次是头部,在腹、足中的表达较低,在胸部微表达。本研究克隆获得西花蓟马气味结合蛋白基因FoccOBP3,明确了其核苷酸、氨基酸序列特征,预测了该蛋白的二级、三维结构,测定了FoccOBP3在西花蓟马中的时空表达,推测该基因可能在西花蓟马气味识别、寄主定位等方面发挥重要作用。

Identifying the olfactory relative proteins in insects and studying their sequence and temporal-spatial expression will lay a basis for throwing light on the chemical communication mechanism between insect and their host plant.A new OBP gene in F.occidentalis was cloned,identified and named as FoccOBP3.The number in GenBank is MT682352.The full length of FoccOBP3 cDNA is 1226 bp.The open reading frame is 432 bp encoding a putative protein of 143 amino acids.The arrangement of typical six-cysteine is C_1-X_(26)-C_(2)-X_(3)-C_(3)-X_(37)-C_4-X_(10)-C_(5)-X_8-C_(6),which completely conforms to the typical OBPs’structural model about six conservative cysteine sites.The deduced amino acid sequence contains 5 lipotropism areas.A significant lipotropy pocket is formed by 62-75 amino acid residues.The molecular mass and isoelectric point of this amino acid sequence are 15.50 kD and 5.24,respectively.There was a putative signal peptide consist of 21 amino acid residues at the N terminus.The amino acid sequence of FoccOBP3 has the highest homolog with Diachasma alloeum DallGOBP(GenBank number is XP015125081.1),Tribolium castaneum TcasPBP(GenBank number is XP015835846.1)and TcasOBP07(GenBank number is EFA04593.1)FoccOBP3 amino acid sequence is clusted into one small group with the amino acid sequence of Nicrophorus vespilloides NvesGOBP(GenBank number is XP017781594.1),suggesting that these genes likely develop from a common ancestral gene.FoccOBP3 protein contains 127 amino acid residues that make upα-helix,58 amino acid residues that formβ-fold and 15 amino acid residues that formβ-corner.The skeleton of FoccOBP3 protein is composed of sixα-helices and the fold back connecting these helices,of which fiveα-helices form a binding pocket.The result of FoccOBP3 expression in different development stages of F.occidentalis indicated that this gene expresses highly in the first instar nymph.The next is in 5-day-old adult female.The expression levels of other development stages are lower very much.In addition,there are also difference in the expression between males and females.The expressions of female on day 1,5 and 15 are higher than those of male at the same developmental stage except on day 10.The result of FoccOBP3 expression in different tissues of F.occidentalis revealed this gene express highly in the antennae.The next is in head.The expression levels of abdomen and leg are lower.The expression level of thorax is the lowest.An odorant binding protein FoccOBP3 from F.occidentalis was cloned in this study.The sequence characteristics of nucleotides and amino acids of FoccOBP3 were clarified.The secondary structure and three-dimensional model of FoccOBP3 were predicted successfully.Temporal-spatial expression pattern of FoccOBP3 was determined,suggesting that FoccOBP3 may play a significant role in odor recognition and host location of F.occidentalis.